Minor groove binder (MGB)-oligonucleotide miRNA antagonists

ABSTRACT

Compositions and methods for inhibiting the actions of non-coding RNAs such as miRNAs and piRNAs are provided. The compositions comprise single or double stranded oligonucleotides conjugated with Minor Groove Binders (“MGBs”). The oligonucleotides can vary in length, can contain nucleotides having one or more modifications, and have regions that are substantially complementary to one or more mature miRNAs or piRNAs.

This application is a divisional application of, and claims priority toU.S. patent application Ser. No. 12/953,098, entitled “Minor GrooveBinder (MGB)-Oligonucleotide miRNA Antagonists,” filed on Nov. 23, 2010,which claims priority to U.S. Provisional Patent Application Ser. No.61/264,380, entitled “minor Groove Binder (MGB)-Oligonucleotide miRNAAntagonist,” filed on Nov. 25, 2009, the entire content of which ishereby incorporated by reference.

BACKGROUND

This invention relates to compositions and methods for inhibiting theactions of non-coding RNAs such as miRNAs and piRNAs.

RNA interference (“RNAi”) is a near-ubiquitous pathway involved inpost-transcriptional gene modulation. The key effector molecule of RNAiis the microRNA (“miRNA” or “miR”). These small, non-coding RNAs aretranscribed as primary miRNAs (“pri-miRNA,”), shown in FIG. 1, andprocessed in the nucleus by Drosha (a Type III ribonuclease) to generateshort hairpin structures called pre-miRNAs. These molecules are thentransported to the cytoplasm and processed by a second nuclease (Dicer)to generate the mature, duplex form of the miRNA which is then capableof being incorporated in the RNA Induced Silencing Complex (“RISC”).Interactions between the mature miRNA-RISC complex and target messengerRNA (“mRNA”) are (in part) mediated by the seed region of the miRNAguide strand (nucleotides 2-7) and lead to gene knockdown by transcriptcleavage and/or translation attenuation.

Tools that enable researchers to understand the roles that miRNAs andmiRNA targets play in disease, cellular differentiation, and homeostasisare invaluable. Such tools include but are not limited to miRNAinhibitors. Classes of miRNA inhibitors have been previously described(see Meister 2004 and Hutvagner 2004). These molecules are singlestranded, range in size from 21-31 nucleotides (“nts”) in length, andcontain O-methyl substitutions at the 2′ position of the ribose ring.Since the original discovery of miRNA inhibitors, multiple designelements have been identified and incorporated to enhance the efficacyof these molecules in a biological setting. For example, it has beendemonstrated that inhibitors that have longer lengths or incorporatesecondary structures (e.g. double stranded inhibitors) exhibit superiorperformance over the shorter 21-31 single stranded nucleotide design(Vermeulen et al. 2007). Other designs include the incorporation oflocked nucleic acids (“LNAs”) (Orom et al. 2006).

SUMMARY

The present invention provides compositions and methods for inhibitingthe actions of non-coding RNAs such as miRNAs and piRNAs. Thecompositions comprise single or double stranded oligonucleotidesconjugated with Minor Groove Binders (“MGBs”). The length of theoligonucleotide portion of the composition can vary considerably.Furthermore, the oligonucleotide can incorporate secondary structuresincluding but not limited to those resulting from hairpins, bulges,and/or mismatches. Preferably the oligonucleotides contain a sequencethat is (at least) substantially complementary (about 70%) to anendogenous mature miRNA or piRNA sequence or sequences.

Without wanting to be bound by theory, the improved performance of miRNAinhibitors likely results from increased binding affinity between theinhibitor and the target molecule. Thus, alternative strategies thatenhance duplex stability or lock the inhibitor-miRNA-RISC complex in amore desirable conformation would further enhance the functionality ofcurrent miRNA inhibitor designs.

Oligonucleotides conjugated to Minor Groove Binders (“MGBs”) can formstable duplexes with complementary sequences (Kutyavin, I. V., et al2000). Though the mechanism behind MGB actions is yet to be fullyunderstood, it has been suggested that MGBs induce conformationalchanges that enhance duplex stability. Similarly, conjugation of MGBs toshort inhibitor molecules is expected to significantly enhance theirpotency over non-MGB inhibitors of similar size.

The minor groove binder component can also vary greatly and include anynumber of structures. Non-limiting examples of the MGB structures can befound in U.S. Pat. Nos. 5,801,155 and 7,582,739, incorporated herein byreference. These MGBs can be conjugated to the 5′ and/or 3′ terminus ofone or more oligonucleotides, or can be associated with one or morenucleotides in the interior of an oligonucleotide.

The compositions disclosed herein are useful in various in vivo or invitro methods for inhibiting miRNA actions. For example, thecompositions can be used in treating a disease or conditioncharacterized by over-expression of a miRNA by administering an optimalamount of an inventive MGB-antagonist against such miRNA.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a general schematic of the RNAi pathway.

FIG. 2 (a) shows a schematic of two MGB configurations (DPI; and CDPI₃moieties) conjugated to oligonucleotides. FIG. 2( b) shows a schematicof exemplary positions in which MGBs can be substituted.

FIG. 3 shows a schematic of the dual luciferase assay.

FIG. 4 a shows the performance of multiple miRNA inhibitor designs onthe let-7c dual luciferase reporter construct. FIG. 4 b shows theperformance of multiple miRNA inhibitor designs on the miR-21 dualluciferase reporter construct.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

I. General

The present invention is directed to compositions and methods forinhibiting RNA interference, including si RNA. piRNA, and miRNA-inducedgene silencing.

The present invention provides compositions and methods for inhibitingthe actions of non-coding RNAs such as miRNAs and piRNAs. Thecompositions comprise single or double stranded oligonucleotidesconjugated with Minor Groove Binders (“MGBs”) through a linker. Theoligonucleotide portion of the molecule can be composed of RNA, DNA, orRNA-DNA hybrids with any of the nucleotides of the above being modifiedor unmodified. The length of the oligonucleotide portion of thecomposition can vary considerably and range from as short as 6nucleotides or base pairs (e.g. the minimal length of the seed region)to as long as 100 nucleotides or base pairs. Furthermore, theoligonucleotide can incorporate secondary structures including but notlimited to those resulting from hairpins, bulges, and/or mismatches.Preferably the oligonucleotides contain a sequence that is (at least)substantially complementary (about 70%) to an endogenous mature miRNA orpiRNA sequence or sequences.

FIG. 1 is a schematic describing the most basic details of the RNAipathway. Endogenous miRNAs are first transcribed as pri-miRNAs thatminimally consist of a hairpin structure with 5′ and 3′ flankingregions. Pri-miRNAs are processed by Drosha to yield pre-miRNAs thatconsist of simplified hairpin structures. Pre-miRNAs are transported outof the nucleus into the cytoplasm where they are further processed byDicer into mature, duplex miRNAs capable of entering RISC and silencinggene expression by either mRNA cleavage or translation attenuation.

II. Definitions

Unless stated otherwise, the following terms and phrases have themeanings provided below:

The term “reporter” or “reporter gene” refers to a gene whose expressioncan be monitored. For example, expression levels of a reporter can beassessed to evaluate the success of gene silencing by substrates of theRNAi pathway.

The term “RNA Induced Silencing Complex,” and its acronym “RISC,” refersto the set of proteins that complex with single-stranded polynucleotidessuch as mature miRNA or siRNA, to target nucleic acid molecules (e.g.,mRNA) for cleavage, translation attenuation, methylation, and/or otheralterations. Known, non-limiting components of RISC include Dicer, R2D2and the Argonaute family of proteins, as well as strands of siRNAs andmiRNAs.

The term “RNA interference” and the term “RNAi” are synonymous and referto the process by which a polynucleotide (a miRNA or siRNA) comprisingat least one polyribonucleotide unit exerts an effect on a biologicalprocess. The process includes, but is not limited to, gene silencing bydegrading mRNA, attenuating translation, interactions with tRNA, rRNA,hnRNA, cDNA and genomic DNA, as well as methylation of DNA withancillary proteins.

The term “gene silencing” refers to a process by which the expression ofa specific gene product is lessened or attenuated by RNA interference.The level of gene silencing (also sometimes referred to as the degree of“knockdown”) can be measured by a variety of means, including, but notlimited to, measurement of transcript levels by Northern Blot Analysis,B-DNA techniques, transcription-sensitive reporter constructs,expression profiling (e.g. DNA chips), qRT-PCR and related technologies.Alternatively, the level of silencing can be measured by assessing thelevel of the protein encoded by a specific gene. This can beaccomplished by performing a number of studies including WesternAnalysis, measuring the levels of expression of a reporter protein thathas e.g. fluorescent properties (e.g. GFP) or enzymatic activity (e.g.alkaline phosphatases), or several other procedures.

The terms “microRNA”, “miRNA”, or “miR” all refer to non-coding RNAs(and also, as the context will indicate, to DNA sequences that encodesuch RNAs) that are capable of entering the RNAi pathway and regulatinggene expression. “Primary miRNA” or “pri-miRNA” represents thenon-coding transcript prior to Drosha processing and includes thestem-loop structure(s) as well as flanking 5′ and 3′ sequences.“Precursor miRNAs” or “pre-miRNA” represents the non-coding transcriptafter Drosha processing of the pri-miRNA. The term “mature miRNA” canrefer to the double stranded product resulting from Dicer processing ofpre-miRNA or the single stranded product that is introduced into RISCfollowing Dicer processing. In some cases, only a single strand of anmiRNA enters the RNAi pathway. In other cases, two strands of a miRNAare capable of entering the RNAi pathway.

The term “mature strand” refers to the sequence in an endogenous miRNAthat is the full or partial reverse complement of (i.e., is fully orpartially complementary to) a target RNA of interest. The terms “maturesequence” or “targeting strand” and “targeting sequence” are synonymouswith the term “mature strand” and are often used interchangeably herein.

The terms “MGB inhibitor,” “MGB miRNA inhibitor,” “MGB antagonist,” and“MGB-oligonucleotide miRNA antagonist” are used interchangeably andrefer to a molecule having an oligonucleotide component conjugated to aminor groove binder (“MGB”) and capable of inhibiting the action of amiRNA or piRNA.

The term “target sequence” refers to a sequence in a target RNA, or DNAthat is partially or fully complementary to the mature strand. Thetarget sequence can be described using the four bases of DNA (A, T, G,and C), or the four bases of RNA (A, U, G, and C).

The term “target RNA” refers to a specific RNA that is targeted by theRNAi pathway, resulting in a decrease in the functional activity of theRNA. In some cases, the RNA target is an mRNA whose functional activityis its ability to be translated. In such cases, the RNAi pathway willdecrease the functional activity of the mRNA by translationalattenuation or by cleavage. In this disclosure, target RNAs are miRNAs,piRNAs, or related molecules whose function can be inhibited by binding.The term “target” can also refer to DNA.

The term “complementary” refers to the ability of polynucleotides toform base pairs with one another. Base pairs are typically formed byhydrogen bonds between nucleotide units in antiparallel polynucleotidestrands. Complementary polynucleotide strands can base pair in theWatson-Crick manner (e.g., A to T, A to U, C to G), or in any othermanner that allows for the formation of duplexes, including the wobblebase pair formed between U and G. As persons skilled in the art areaware, when using RNA as opposed to DNA, uracil rather than thymine isthe base that is considered to be complementary to adenosine. However,when a U is denoted in the context of the present invention, the abilityto substitute a T is implied, unless otherwise stated.

The term “duplex” refers to a double stranded structure formed by twocomplementary or substantially complementary polynucleotides that formbase pairs with one another, including Watson-Crick base pairs and U-Gwobble pairs that allow for a stabilized double stranded structurebetween polynucleotide strands that are at least partiallycomplementary. The strands of a duplex need not be perfectlycomplementary for a duplex to form, i.e., a duplex may include one ormore base mismatches. In addition, duplexes can be formed between twocomplementary regions within a single strand (e.g., a hairpin).

The term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide or modified form thereof, as well as an analogthereof. Nucleotides include species that comprise purines, e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs, aswell as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs. Nucleotide analogs include nucleotides havingmodifications in the chemical structure of the base, sugar and/orphosphate, including, but not limited to, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atcytosine exocyclic amines, and substitution of 5-bromo-uracil; and2′-position sugar modifications, including but not limited to,sugar-modified ribonucleotides in which the 2′-OH is replaced by a groupsuch as an H. OR, R, halo, SH, SR. NH.sub.2, MIR, NR.sub.2, or CN,wherein R is an alkyl moiety. Nucleotide analogs are also meant toinclude nucleotides with bases such as inosine, queuosine, xanthine,sugars such as 2′-methyl ribose, non-natural phosphodiester linkagessuch as in ethylphosphonates, phosphorothioates and peptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications that cancomprise nucleotides that are modified with respect to the base moietiesinclude but are not limited to, alkylated, halogenated, thiolated,aminated, amidated, or acetylated bases, individually or in combination.More specific examples include, for example, 5-propynyluridine,5-propynytcytidine, 6-methyladenine, 6-methylguanine,N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any O- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoai nleotides, and alkylcarbonylalkylatednucleotides. Modified nucleotides also include those nucleotides thatare modified with respect to the sugar moiety, such as by containing a2′-0,4′-C methylene bridge, as well as nucleotides having sugars oranalogs thereof that are not ribosyl. For example, the sugar moietiesmay be, or be based on, mannoses, arabinoses, glucopyranoses,galactopyranoses, 4′-thioribose, and other sugars, heterocycles, orcarbocycles.

The term nucleotide is also meant to include what are known in the artas universal bases. By way of example, universal bases include, but arenot limited to, 3-nitropyrrole, 5-nitroindole, or nebularine. The term“nucleotide” is also meant to include the N3′ to P5′ phosphoramidate,resulting from the substitution of a ribosyl 3′-oxygen with an aminegroup. Further, the term nucleotide also includes those species thathave a detectable label, such as for example a radioactive orfluorescent moiety, or mass label attached to the nucleotide.

III. Description of the Embodiments

In one embodiment, a MGB miRNA inhibitor comprises an oligonucleotidecomponent and an MGB-linker combination, with the linker having fromabout 3 to 100 main chain atoms, selected from C, O, N, S, P and Si. Thelinker can be a trivalent linker, a branched aliphatic chain, aheteroalkyl chain, one or more substituted ring structures, orcombinations thereof. In one preferred embodiment, the inhibitorcomprises a single stranded oligonucleotide that (1) can vary in lengthbetween 6-100 nucleotides in length, (2) has regions that aresubstantially complementary to one or more mature miRNAs or piRNAs orportions of mature miRNAs or piRNAs, and (3) is conjugated to one ormore minor groove binders (MGB) through a linker. Preferably, themolecule comprises an MGB that is DPI; or CDPI₃.

Another embodiment pertains to a method for modulating gene expression;the method comprising introducing into a cell, in vitro or in vivo, anMGB miRNA inhibitor at a concentration such that the function of atarget nucleic acid, preferably a miRNA or piRNA, is inhibited.

Another embodiment pertains to a method of treating a disease orcondition that results from mis-expression of a gene or expression of agene that has an undesirable function. The method comprisesadministering sufficient amounts of one or more MGB-miRNA inhibitorsdisclosed herein, with or without a suitable pharmaceutical carrier, toa patient suspected of having such a disease or condition.

Preferably one or more nucleotides of the oligonucleotide portion of theMGB inhibitor are modified. The preferred modification is an O-alkylmodification of the T carbon of the ribose ring of some or all of thenucleotides. Such modifications greatly enhance the affinity of themolecule for the target nucleic acid. That said, the MGB inhibitors ofthe invention exhibit multiple improvements over simple, modified singlestranded inhibitors of equivalent length. Most importantly, MGBinhibitors exhibit enhanced potency of silencing.

Multiple design elements are taken into consideration when developingthe highly functional MGB inhibitors described herein. These include (1)single stranded vs. multi-stranded designs, (2) oligonucleotide length.(3) oligonucleotide content (in the targeting portion and/ornon-targeting portions of the oligonucleotide). (4) chemicalmodifications of the oligonucleotides. (5) the type of MGB conjugate,(6) the position of the MGB conjugate on the oligonucleotide, and (7)the type of linker that is used to associate the MGB moiety to theoligonucleotide. The following descriptions address each of theseelements in greater detail.

A. Inhibitor Design

Inhibitor designs that are compatible with the MGB enhancements includeboth single stranded and multi-stranded designs. For example, theoligonucleotide portion of the inhibitor can be single stranded, fullydouble stranded, or a combination of single and double stranded regions(e.g., containing hairpin loop(s)). Additional details on MGB-compatibleinhibitor designs can be found in WO2007/095387.

B. Oligonucleotide Length

The length of the oligonucleotide that is associated with an MGB canvary depending on a number of factors including the length of theendogenous miRNA being targeted by the molecule and the desired designattributes of the inhibitor. Mature miRNAs can vary in length from about18 bp to 28 base pairs. As such, in one embodiment, the length of theoligonucleotide conjugated to the MGB is the reverse complement to themature strand of the miRNA being targeted. Reverse complements for allthe known miRNAs can be determined from miRNA mature strand sequenceswhich can be found in miRBase (microrna.sanger.ac.uk) which ismaintained by the Sanger Institute. It should be noted that the list ofsequences available in miRBase is predicted to increase as the number ofmiRNA sequences in all species expands. As such, the number of potentialsequences that MGB-inhibitors can target is expected to grow.

In other instances, studies have shown that the performance of non-MGBinhibitors increases with increasing length (see Vermeulen et al 2007).As such, in another embodiment the MGB inhibitors can include sequencesthat flank the sequence which is the reverse complement of the miRNAbeing targeted. The length of these sequences vary greatly (5-100nucleotides on the 5′ and/or 3′ end) and can comprise (1) the reversecomplement of sequences flanking the mature sequence in the pre-miRNA orpri-miRNA, or (2) sequences partially related or unrelated to thereverse complement of the pre-miRNA or pri-miRNA.

C. The MGB component of MGB miRNA inhibitors

Multiple MGBs can be incorporated into the MGB-inhibitor design. In onenon-limiting example. DPI₃ and CDPI₃ minor groove binder ligands can beattached to the oligonucleotide in any number of orientations using awide range of linker chemistries known in the art. Preferably, the minorgroove binders are conjugated to either the 3′ or 5′ end of the strandof the inhibitor that is the reverse complement to, e.g., the targetingstrand of the target miRNA.

FIG. 2 (a) is a schematic of two MGB configurations (DPI₃, and CDPI₃,moieties) conjugated to oligonucleotides. FIG. 2( b) is a schematicshowing positions in which MGBs can be substituted. In FIG. 2( b), W isa linker having from about 3 to 100 main chain atoms, selected from C,O, N, S, P and Si. Generally, W represents a trivalent linker, abranched aliphatic chain, a heteroalkyl chain, one or more substitutedring structures, or combinations thereof. [A-B]_(n) represents a nucleicacid oligomer (e.g., DNA. RNA, PNA or any combination thereof, includingthose with modified bases and sugars) wherein A represents a sugarphosphate backbone, modified sugar phosphate backbone, locked nucleicacid backbone, peptidic backbone or a variant thereof used in nucleicacid preparation; and B represents a nucleic acid base, a modified baseor a base analog as described in more detail below. The subscript n isan integer of from about 3 to about 100, preferably 6 to about 50 andmore preferably 8 to about 20. The symbols R_(a), R_(b), R_(c), R_(d),R_(e) and R_(f) represent substituents selected from H, halogen,(C₁-C₈)alkyl, OR_(g), N(R_(g))₂, N⁺(R_(g))₃, SR_(g), COR_(g), CO₂R_(g),CON(R_(g))₂, (CH₂)_(m)SO₃ ⁻; (CH₂)_(m)CO₂ ⁻; (CH₂)_(m)OPO₃ ⁻², andNHC(O)(CH₂)_(m)CO₂ and esters and salts thereof, wherein each R_(g) isindependently H or (C₁-C₈)alkyl, and the subscript m is an integer offrom 0 to 6. The symbol R_(h) and R_(w) represents H or a group(typically the vestige of a linking group used in solid phase synthesis)having from 1-30 atoms selected from C, N, O, P, and S which is eithercyclic, acyclic, or a combination thereof, and having additionalhydrogen atoms to fill the available valences. Additional examples ofsubstituents can be found in U.S. Patent Application Publication No.2005/01 18623.

As stated above, the substituent A can include a deoxyribofuranosephosphate backbone or a ribofuranose phosphate backbone. In preferredembodiments the ribofuranose is substituted as shown below:

wherein Rz is —OR^(aa) where R^(aa) is —O-Alkyl₁₋₁₂,—(CH₂)_(n)O-Alkyl₁₋₁₂ where n is 1 to 6, halogen, or —CF₃; and B is anormal base or a modified base as defined above or in U.S. Pat. No.7,045,610. The phosphate backbone of the modified oligonucleotidesdescribed above can also be modified so that the oligonucleotidescontain phosphorothioate linkages and/or methylphosphonates and/orphosphoroamidates (Chen et al., Nucl, Acids Res., 23:2662-2668 (1995)).Combinations of oligonucleotide linkages in MB-oligonucleotideconjugates are also within the scope of the present invention. Stillother backbone modifications are known to those of skill in the art.

Some minor groove binders contain different repeating units. Preferredminor groove binders are:

wherein the subscript m is an integer of from 2 to 5; the subscript r isan integer of from 2 to 10; and each R^(a) and R^(b) is independently alinking group to the oligonucleotide (either directly or indirectlythrough a quencher), H, —OR^(c), —NR^(c)R^(d), —COOR^(c) or—CONR^(c)R^(d), wherein each R^(c) and R^(d) is selected from H,(C₂-C₁₂)heteroalkyl, (C₃-C₁₂)heteroalkenyl, (C₃-C₁₂)heteroalkynyl,(C₁-C₁₂)alkyl, (C₂-C₁₂alkenyl, (C₂-C₁₂)alkynyl, aryl(C₁-C₁₂)alkyl andaryl, with the proviso that one of R^(a) and R^(b) represents a linkinggroup to ODN or fluorophore. In an additional embodiment each of therings in each structure can contain one or more additional substitutionsselected from H, halogen, (C₁-C₁₂alkyl, OR_(g), N(R_(g))₂, N⁺(R_(g))₃,SR_(g), COR_(g), CO₂R_(g), CON(R_(g))₂, (CH₂)_(m)SO₃ ⁻, (CH₂)_(m)CO₂ ⁻,(CH₂)_(m)OPO₃ ⁻², and NHC(O)(CH₂)_(m)CO₂ ⁻, AsO—₃ ²⁻, and esters andsalts thereof, wherein each R_(g) is independently H or (C₁-C₈)alkyl,and the subscript m is an integer of from 0 to 6. Additional detailsregarding these structures can be found in U.S. Patent ApplicationPublication Nos. 2004/32665 and 2006/0229441.

Other Minor Groove Binders of interest have been disclosed in U.S. Pat.No. 6,312,894. In one group of embodiments, the MGB is selected from thegroup consisting of CC1065, lexitropsins, distamycin, netropsin,berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole,stilbamidine, 4,4′-diacetyldiphenylurea bis(guanylhydrazone) (DDUG), andpyrrolo[2,1-c][1,4]benzodiazepines or any of their analogs.

D. Oligonucleotide Content of an MGB Inhibitor

The oligonucleotide portion of the MGB inhibitors can consist of RNA,DNA, RNA-DNA hybrids and modifications of the same. In general, thesequence of some portion of each inhibitor is designed to be the reversecomplement of a given miRNA expressed by the cell of interest.Alternatively, in cases where a miRNA is but one representative of afamily of related sequences (e.g., let-7 family), the oligonucleotideportion of the MGB-inhibitor can comprise the reverse complement of,e.g., one family member, but have one or more bulges or base pairmismatches when aligned with other members in the miRNA family. As such,preferably the oligonucleotide portion of the MGB inhibitor is at least70-80% complementarity to a target miRNA. More preferably theoligonucleotide portion of the MGB inhibitor has at least 80-99%complementarity to a target miRNA. And most preferably, theoligonucleotide portion of the MGB inhibitor has 100% complementarity toa target miRNA.

The nucleotides of the oligonucleotide portion of MGB inhibitors cancontain a variety of chemical modifications that enhance the resilienceagainst nuclease action, the deliverability of the molecule to cells,specificity, or the stability of the duplex (i.e. between the targetmiRNA and the oligonucleotide portion of the MGB inhibitor). Chemicalmodifications that provide these desired traits are well known in theart and include but are not limited to alterations/modifications of thebase, the internucleotide linkage, as well as the sugar residue of theoligonucleotide. Some preferred modifications are listed below and aredescribed in U.S. Pat. No. 7,045,610. These include 2′-O-alkylmodifications (e.g., 2′-O-methyl), 2′ halogen modifications (e.g., 2°F.), 5′ and/or 3′ cholesterol modifications and more. Furthermore. MGBinhibitors can include additional modifications that provide beneficialattributes to the molecule(s). Thus, for instance, MGB inhibitors can befurther modified with, e.g., fluorescent dyes as well as, e.g.,cholesterol modifications to enhance visualization and delivery of theMGB-inhibitors, respectively.

An example of a modification that can be associated with the polymericbackbone of the MGB antagonists is shown below:

wherein Rz is —H and R═—C≡C—CH₂CH₂OH. This structure is also known asSuper A.

E. Method of Introducing and Detecting the Effects of MGB-Inhibitors

The inhibitors of the present invention can be used in vitro, oradministered to a cell or an animal including humans by any method knownto one skilled in the art. For example, the molecules of the inventionmay be passively delivered to cells. Passive uptake of an inhibitor canbe modulated, for example, by the presence of a conjugate such as apolyethylene glycol moiety or a cholesterol moiety, or any otherhydrophobic moiety associated with the 5′ terminus, the 3′ terminus, orinternal regions of the oligonucleotide. Alternatively, passive deliverycan be modulated by conjugation of a ligand that is taken up by a cellthrough receptor mediated endocytosis. Other methods for inhibitordelivery include, but are not limited to, transfection techniques (usingforward or reverse transfection techniques) employing DEAE-Dextran,calcium phosphate, cationic lipids/liposomes, microinjection,electroporation, immunoporation, and coupling of the inhibitors tospecific conjugates or ligands such as antibodies, peptides, antigens,or receptors.

The method of assessing the level of inhibition is not limited. Thus,the effects of any inhibitor can be studied by one of any number of arttested procedures including but not limited to Northern analysis, RTPGR, expression profiling, and others. In one preferred method, a vectoror plasmid encoding reporter whose protein product is easily assayed ismodified to contain the target site (reverse complement of the maturemiRNA, piRNA, or siRNA) in the 5′ UTR. ORF, or 3′ UTR of the sequence.Such reporter genes include alkaline phosphatase (AP), betagalactosidase (LacZ), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), variants of luciferase (Luc), and derivativesthereof. In the absence of the inhibitor, endogenous (or exogenouslyadded) miRNAs target the reporter mRNA for silencing (either bytranscript cleavage or translation attenuation) thus leading to anoverall low level of reporter expression. In contrast, in the presenceof the inhibitors of the invention, miRNA (piRNA, or siRNA) mediatedtargeting is suppressed, thus giving rise to a heightened level ofreporter expression. Preferred reporter constructs include thepsiCHECK-2 dual luciferase reporter (Promega).

IV. Applications

The inhibitors of the present invention may be used in a diverse set ofapplications, including basic research. For example, the presentinvention may be used to validate whether a miRNA or target of a miRNAis a target for drug discovery or development. Inventive inhibitors thatinhibit a particular miRNA or a group of miRNAs are introduced into acell or organism and said cell or organism is maintained underconditions that allow for specific inhibition of the targeted molecule.The extent of any decreased expression or activity of the target is thenmeasured, along with the effect of such decreased expression oractivity, and a determination is made that if expression or activity isdecreased, then the target is an agent for drug discovery ordevelopment. In this manner, phenotypic effects can be associated withinhibition of particular target of interest, and in appropriate casestoxicity and pharmacokinetic studies can be undertaken and therapeuticpreparations developed.

The molecules of the invention can be used to inhibit single or multipletargets simultaneously. Knockdown of multiple targets can take place byintroducing pools of inhibitors targeting different molecules. Previousinhibitor designs lacked potency and as such, required highconcentrations to partially inhibit e.g. a single miRNA. Introduction ofpools of inhibitors using previous designs would require excessivelyhigh concentrations that can be cytotoxic. In contrast, the enhancedpotency of the molecules of the invention enables users to inhibit oneor more specific targets at concentrations that preserve the overallfunctionality of the RNAi pathway with minimal non-specific effects.

Because the inhibitors of the invention act independent of the cell typeor species into which they are introduced, the present invention isapplicable across a broad range of organisms, including but not limitedplants, animals, protozoa, bacteria, viruses and fungi. The presentinvention is particularly advantageous for use in mammals such ascattle, horse, goats, pigs, sheep, canines, birds, rodents such ashamsters, mice, and rats, and primates such as, gorillas, chimpanzees,and humans.

The present invention may be used advantageously with diverse celltypes, including but not limited to primary cells, germ cell lines andsomatic cells. For example, the cell types may be embryonic cells,oocytes, sperm cells, adipocytes, fibroblasts, myocytes, cardiomyocytes,endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes,macrophages, neutrophils, eosinophils, basophils, mast cells,leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts,osteoclasts, hepatocytes and cells of the endocrine or exocrine glands.Importantly, the present invention can be used to inhibit a broad rangeof miRNA, piRNA, and siRNAs including but not limited to (1) miRNA andpiRNAs of the human genome implicated in diseases such as diabetes,Alzheimer's, and cancer, and (2) those associated with the genomes ofpathogens (e.g. pathogenic viruses).

Still further, the present invention may be used in RNA interferenceapplications, such as diagnostics, prophylactics, and therapeuticsincluding use of the compositions in the manufacture of a medicament inanimals, preferably mammals, more preferably humans in the treatment ofdiseases. In particular, the agents of the invention can be used toreverse the action of siRNAs, miRNAs, or piRNAs that are being used astherapeutic agents.

In the case of therapeutic or prophylactic purposes, dosages ofmedicaments manufactured in accordance with the present invention mayvary from micrograms per kilogram to hundreds of milligrams per kilogramof a subject. As is known in the art, dosage will vary according to themass of the mammal receiving the dose, the nature of the mammalreceiving the dose, the severity of the disease or disorder, and thestability of the medicament in the serum of the subject, among otherfactors well known to persons of ordinary skill in the art. For theseapplications, an organism suspected of having a disease or disorder thatis amenable to modulation by manipulation of a particular target nucleicacid of interest is treated by administering inhibitors of theinvention. Results of the treatment may be ameliorative, palliative,prophylactic, and/or diagnostic of a particular disease or disorder.

Therapeutic or prophylactic applications of the present invention can beperformed with a variety of therapeutic compositions and methods ofadministration. Pharmaceutically acceptable carriers and diluents areknown to persons skilled in the art. Methods of administration to cellsand organisms are also known to persons skilled in the art. Dosingregimens, for example, are known to depend on the severity and degree ofresponsiveness of the disease or disorder to be treated, with a courseof treatment spanning from days to months, or until the desired effecton the disorder or disease state is achieved. Chronic administration ofinhibitors of the invention may be required for lasting desired effectswith some diseases or disorders. Suitable dosing regimens can bedetermined by, for example, administering varying amounts of one or moreinhibitors in a pharmaceutically acceptable carrier or diluent, by apharmaceutically acceptable delivery route, and amount of drugaccumulated in the body of the recipient organism can be determined atvarious times following administration. Similarly, the desired effectcan be measured at various times following administration of theinhibitor, and this data can be correlated with other pharmacokineticdata, such as body or organ accumulation. Those of ordinary skill candetermine optimum dosages, dosing regimens, and the like. Those ofordinary skill may employ EC₅₀ data from in vivo and in vitro animalmodels as guides for human studies.

The inhibitors of the invention can be administered in a cream orointment topically, an oral preparation such as a capsule or tablet orsuspension or solution, and the like. The route of administration may beintravenous, intramuscular, dermal, subdermal, cutaneous, subcutaneous,intranasal, oral, rectal, by eye drops, by tissue implantation of adevice that releases the inhibitor at an advantageous location, such asnear an organ or tissue or cell type harboring a target nucleic acid ofinterest.

The foregoing embodiments are presented in order to aid in anunderstanding of the present invention and are not intended, and shouldnot be construed, to limit the invention in any way. All alternatives,modifications and equivalents that may become apparent to those ofordinary skill upon reading this disclosure are included within thespirit and scope of the present invention.

EXAMPLES

The following examples are provided to illustrate, but not to limit, thepresently claimed invention.

Example 1 Preparation of MGB Inhibitors

DPI₃-modified oligonucleotides were prepared using DPI₃; solid DNAsynthesis support as described in U.S. Pat. No. 7,381,818. The followingsteps were undertaken in the preparation of CDPI₃-modifiedoligonucleotides.

1. HPLC purification and salt exchange of amine-modified oligos.Amine-modified oligonucleotides (0.2-1 μmol synthesis scale) weredissolved in 0.1 M TEAB (triethylammonium bicarbonate) buffer to ˜1 mland chromatographed on a Luna C18 (10 μm) 4.6×250 mm column (Phenominex)eluting with a gradient of CH₃CN in 0.1 M TEAB buffer. The productcontaining fraction were collected and dried in a SpeedVac concentratoruntil dry pellets were obtained.

2. CDPI₃ conjugation reaction. To each tube containing an amine-modifiedoligonucleotide (0.2-1 μmol initial DNA synthesis scale) was added asolution of 1 mg of CDPI₃TFP ester shown below (and also furtherdescribed in U.S. Pat. No. 5,801,155) and 2 ml TEA in 80 μl of DMSO. Thetubes were gently swirled to dissolve the solids. The conjugationreactions were allowed to proceed for 5-18 hrs.

3. Conjugate purification. The reactions were diluted with 2 ml of 0.1 MTEAB buffer, loaded onto Luna C18 column and eluted with a gradient(8-40% of CH₃CN) in 0.1 M TEAB buffer. The product containing fractionwere collected and dried in a SpeedVac concentrator until dry pelletswere obtained.

Example 2 Assay for Assessing miRNA Inhibitor Function

For most of the experiments reported, quantitation of the level ofinhibition was performed using the dual luciferase reporter system,psiCheck 2 (Promega). FIG. 3 is a schematic of the dual luciferaseassay. The dual luciferase reporter contains both (1) the Fluc reporterand (2) an Rluc reporter containing a miRNA target site (miR-X targetsite) in the 3′ UTR, In instances where (1) a non-targeting miRNAinhibitor control is present and (2) an endogenous miRNA (miRNA-X)capable of targeting the Rluc construct is expressed, the relative ratioof Rluc to Flue is suppressed. In contrast, when a miRNA inhibitorcapable of targeting the endogenously expressed miRNA (miRNA-X) is alsopresent, the ability of the miRNA to target the Rluc construct issuppressed and therefore the Rluc to Flue ratio is increased.

Briefly, the psiCheck plasmid encodes for two variants of luciferase,Renilla and Firefly. Target sequences were inserted into the multiplecloning site of the 3′ UTR of the Renilla luciferase gene, thus allowingthe Firefly sequence to be used as an internal control. To determine thepracticality of different inhibitor designs, the oligonucleotide(s) ofthe invention and the modified psiCheck 2 plasmid were co-transfectedinto cells (100 ng of reporter DNA per well, 25-100 nM inhibitor,lipid=DharmaFECT Duo, Thermo Fisher Scientific). Twenty-four toninety-six hours later cells were lysed and the relative amounts of eachluciferase was determined using the Dual Glo Assay (Promega). For allexperiments, unless otherwise specified, no significant levels ofcellular toxicity were observed.

Firefly and Renilla luciferase activities were measured using theDual-Glo™ Luciferase Assay System (Promega, Cat.# E2980) according tomanufacturer's instructions with slight modification. When lysing cells,growth media was aspirated from the cells prior to adding 50 μL offirefly luciferase substrate and 50 μL Renilla luciferase substrate.

The Luciferase assays were all read with a Wallac Victor² 1420multilabel counter (Perkin Elmer) using programs as recommended by themanufacturers.

Experimental Design and Data Analysis: All treatments were run intriplicate. In addition, each experimental treatment with a reporterplasmid was duplicated with the psiCHECK™-2 control plasmid (no insert).To account for non-specific effects on reporter plasmids, experimentalresults are expressed as a normalized ratio (Rluc/Fluc)_(norm): theratio of Renilla luciferase expression to firefly luciferase expressionfor a given miRNA reporter plasmid (Rluc/Fluc)_(miRNA) divided by the(Rluc/Fluc)_(control) ratio for the identically treated psiCHECK™-2reporter plasmid. The maximum values obtained from the reporter plasmidvary due to sequence. Ideally, values around 1 indicate low miRNAfunction, while values close to zero indicate high miRNA function. Dataare reported as the average of the three wells and the error bars arethe standard deviation of the three (Rluc/Fluc)_(miRNA) ratios from theexperimental treatment, scaled by the normalizing factor (the average of(Rluc/Fluc)_(control)). While ratios do not follow a normaldistribution, the standard deviation values give a good sense of thevariability of the data.

In cases where values between different miRNA reporter plasmids arecompared, the maximum normalized (Rluc/Fluc)_(norm) ratio was used as anadditional scaling factor so that all reporters have a maximum ofapproximately 1. The additional scaling was performed for ease ofcomparison and does not affect the results.

Cell Culture. HeLa cells were grown under standard conditions andreleased from the solid support by trypsinization. For most assays,cells were diluted to 1×10⁵ cells/ml, followed by the addition of 100 μLof cells/well. Plates were then incubated overnight at 37° C., 5% CO*.

Example 3 Testing Different Designs of Minor Groove Binder Inhibitors

Using the Dual Luciferase Assay described above, a number ofoligonucleotides, modified oligonucleotides and MGB-oligonucleotideconjugates were evaluated as inhibitors of miRNA function. The targetsequences inserted into the 3′ UTR of Rluc were Let7cTcomp from Table 1(for Let7c) and _miR21Tcomp from Table 2 (for miR-21). The sequences forinhibitors of let-7c miRNA and miR-21 miRNA are shown in Table 1 andTable 2 respectively. In Tables 1 and 2, the presence of a2′-O-Methylribofuranose sugar in an oligonucleotide is shown in bolditalics. The presence of a Super A modified base is shown with alowercase letter “a.”

TABLE 1 Sequences of let7c and let7c inhibitors Inhibitor Abrevia- tionSequence Description Let7c 5′-UGAGGUAGUAGGUUGUAUGGUU-3′RNA mature strand (Seq ID No: 1) Let7cT 5′-TGAGGTAGTAGGTTGTATGGTT-3′DNA equivalent (Seq ID No: 2) mature strand Let7cTcomp5′-AACCATACAACCTACTACCTCA-3′ DNA complement (Seq ID No: 3) 2′Omet 5′-

-3′ Is a 2′-OMeRNA (Seq ID No: 4) DNA 5′-AACCATACAACCTACTACCTCA-3′Is a DNA equivalent (Seq ID No: 3) 3MGB-DNA5′-AACCATACAACCTACTACCTCA-MGB-3′ MGB is DPI₃ ligand (Seq ID No: 5)superA DNA 5′-AaCCaTaCAaCCTaCTaCCTCA-3′ “a” is Super A (Seq ID No: 6)5MGB-DNA 5′-MGB-AACCATACAACCTACTACCTCA-3′ (Seq ID No: 7) SuperA 5′-

-3′ “a” is Super A DNA; 2′Omet (Seq ID No: 8) Other bases 2′-OMe 5′MGB5′-MGB-

-3′ 5′-MGB-2′-OMe- 2′Omet (Seq ID No: 9) RNA 3′MGB- 5′-

-MGB-3′ 3′-MGB-2′-OMe- 2′Omet (Seq ID No: 10) RNA

TABLE 2 Sequences of mir21 and mir 21 inhibitors Inhibitor AbreviationSequence Description mir21 5′-UAGCUUAUCAGACUGAUGUUGA-3′RNA mature strand (Seq ID No: 11) mir21T 5′-TAGCTTATCAGACTGATGTTGA-3′DNA equivalent mature (Seq ID No: 12) strand mir2lTcomp5′-TCAACATCAGTCTGATAAGCTA-3′ DNA complement (Seq ID No: 13) 2′Omet 5′-

-3′ Is a 2′-OMeRNA (Seq ID No: 14) DNA 5′-TCAACATCAGTCTGATAAGCTA-3′Is a DNA equivalent (Seq ID No: 13) 3MGB-DNA5′-TCAACATCAGTCTGATAAGCTA-MGB-3′ MGB is DPI₃ ligand (Seq ID No: 15)superA DNA 5′-TCaaCaTCaGTCTGaTAaGCTA-3′ “a” is Super A is a 2′-(Seq ID No: 16) deoxyribonucleotide 5MGB-DNA5′-MGB-TCAACATCAGTCTGATAAGCTA-3′ MGB is DPI₃ligand (Seq ID No: 17)SuperA 5′-

-3′ “a” is Super A DNA; 2′Omet (Seq ID No: 18) Other bases 2′-OMe 5′MGB5′MGB-

-3′ 5′-MGB-2′-OMe-RNA 2′Omet (Seq ID No: 19) 3′MGB- 5′-

A-3′ 3′-MGB-2′-Ome-RNA 2′Omet (Seq ID No: 20)

FIG. 4 shows the performance of the multiple miRNA inhibitor designs.Inhibitors of different designs were introduced into cells together withthe appropriate (let-7c or miR-21) dual luciferase reporter construct.Controls consisted of untreated cells (none) or cells treated withsimple, 2′-O methyl modified reverse complement inhibitor molecules(2″-Omet).

The performance of let-7c inhibitors is shown in FIG. 4 a. A baselineratio of Rluc/Fluc was obtained in the absence of any inhibitor molecule(see “none”). Compared to the untreated control (none) 2′-O methylmodified single stranded inhibitors (2′Omet) showed an increase in theRluc/Fluc ratio, indicating that this design is capable of providingsome level of let-7c inhibition. The “DNA”, “3′-MGB-DNA”. “5′-MGB-DNA”.“Super A substituted DNA” (which refers to a 2′-deoxyribonucleosidedisclosed in U.S. Pat. No. 7,045,610) and the chimera-“Super A-2′Omet”showed baseline levels of inhibition similar to the untreated controls,suggesting that these design configurations were incapable of inhibitinglet-7c function. However, while the “3′-MGB-2′-OMet” induced similarlevels of inhibition as 2′-Omet, the 22-mer “5′-MGB-2′-OMe” inducedroughly 3.4 fold greater levels of inhibition as the 2′-Omet design.Results from a parallel experiment performed on miR-21 show very similarresults (see FIG. 4 b). The 5′-MGB-2′-OMet and 3′-MGB-2′-OMet inhibitorconfigurations exhibited roughly 10× and 3× performance improvementsover the 2*-Omet design, respectively.

REFERENCES U.S. Patent Documents

-   U.S. Pat. No. 5,801,155-   U.S. Pat. No. 6,312,894-   U.S. Pat. No. 7,045,610-   U.S. Pat. No. 7,381,818-   U.S. Pat. No. 7,582,739-   U.S. Patent Application Publication No. 2004/32665-   U.S. Patent Application Publication No. 2005/0118623-   U.S. Patent Application Publication No. 2006/0229441

International Patent Documents

-   PCT Application Publication No. WO2007/095387

Other Publications

-   Chen et al., Nucl. Acids Res. 23:2662-2668 (1995)-   Hutvagner, G. et al. (2004) “Sequence-specific inhibition of small    RNA function.” PLoS Biol. April; 2(4):E98-   Kutyavin, I. V., et al, (2000) “3′-Minor groove binder-DNA probes    increase sequence specificity at PCR extension temperatures.” NAR.    28(2):655-661-   Meister, G. et al. (2004) “Sequence-specific inhibition of microRNA-    and siRNA-induced RNA silencing.” RNA 10(3):544-50-   Orom et al, (2006) “LNA-modified oligonucleotides mediate specific    inhibition of microRNA function” Gene 372:137-141-   Vermeillen et al. RNA, 2007 13(5):723-30

What is claimed is:
 1. A method of inhibiting miRNA activity in vitro orin vivo comprising introducing an inhibitor composition to a location invitro or in vivo where miRNA activity exists; wherein the inhibitorcomposition comprises: an oligonucleotide; and a minor groove binder(MGB), wherein the oligonucleotide comprises nucleotides, wherein allnucleotides of the oligonucleotide comprise ribofuranose having thefollowing structure:

wherein Rz is —OCH₃, and wherein B is a normal base or a modified base,wherein the nucleotides are linked by natural phosphodiester linkages,and wherein the MGB is conjugated to the 5′ end of the oligonucleotide.2. A method of treating a condition characterized by over-expression ofmiRNA comprising administering an inhibitor composition to a subject ata concentration sufficient to inhibit the action of said miRNA, whereinthe inhibitor composition comprises: an oligonucleotide; and a minorgroove binder (MGB) wherein the oligonucleotide comprises nucleotides,wherein all nucleotides of the oligonucleotide comprise ribofuranosehaving the following structure:

wherein Rz is —OCH₃, and wherein B is a normal base or a modified base,wherein the nucleotides are linked by natural phosphodiester linkages,and wherein the MGB is conjugated to the 5′ end of the oligonucleotide.3. The method of claim 1 wherein the inhibitor composition furthercomprising a linker through which the MGB is attached to theoligonucleotide.
 4. The method of claim 1 wherein the oligonucleotide issubstantially complementary to an endogenous mature miRNA.
 5. The methodof claim 1 wherein the oligonucleotide comprises one or more modifiedbases or universal bases.
 6. The method of claim 1, wherein the MGBcomprises a structure selected from the following group:

wherein the subscript m is an integer of from 2 to 5; the subscript r isan integer of from 2 to 10; and each R^(a) and R^(b) is independently alinker to the oligonucleotide, a linker to a flurophore, H, —OR^(c),—NR^(c)R^(d), —COOR_(c) or —CONR^(c)R^(d), wherein each R^(c) and R^(d)is selected from H, (C₂-C₁₂)heteroalkyl, (C₃-C ₁₂)heteroalkenyl,(C₃-C₁₂)heteroalkynyl, (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl,aryl(C₁-C₁₂)alkyl and aryl, wherein one of R_(a) and R^(b) is a linkerto the oligonucleotide or to a fluorophore.
 7. The method of claim 1wherein the inhibitor composition comprises one of the followingstructures:

wherein W is a linker having from about 3 to 100 main chain atoms,selected from C, O, N, S, P and Si; [A-B]_(n) is an oligonucleotidewherein A represents a sugar phosphate backbone, modified sugarphosphate backbone, locked nucleic acid backbone, peptidic backbone or avariant thereof used in nucleic acid preparation. wherein W is connectedto the 5′ end of [A-B]_(n), and wherein [A-B]_(n) comprises nucleotidescomprising ribofuranose having the following structure:

wherein Rz is —OCH₃, and B is a base, and wherein the nucleotides arelinked by natural phosphodiester linkages; n is an integer of from about3 to about 100; R_(a), R_(b), R_(c), R_(d), R_(e) and R_(f) aresubstituents selected from H, halogen, (C₁-C₈)alkyl, OR_(g), N(R_(g))₂,N⁺(R_(g))₃, SR_(g), COR_(g), CO₂R_(g), CON(R_(g))₂, (CH₂)_(m)SO₃ ⁻,(CH₂)_(m)CO₂ ⁻, (CH₂)_(m)OPO₃ ⁺², and NHC(O)(CH₂)_(m)CO₂ ⁺, and estersand salts thereof, wherein each R_(g) is independently H or(C₁-C₈)alkyl, and the subscript m is an integer of from 0 to 6; R_(h)and R_(w) are H or a group having from 1 to 30 atoms selected from C, N,O, P, and S and which is cyclic, acyclic, or a combination thereof. 8.The method of claim 2 wherein the inhibitor composition furthercomprising a linker through which the MGB is attached to theoligonucleotide.
 9. The method of claim 2 wherein the oligonucleotide issubstantially complementary to an endogenous mature miRNA.
 10. Themethod of claim 2 wherein the oligonucleotide comprises one or moremodified bases or universal bases.
 11. The method of claim 2, whereinthe MGB comprises a structure selected from the following group:

wherein the subscript m is an integer of from 2 to 5; the subscript r isan integer of from 2 to 10; and each R^(a) and R^(b) is independently alinker to the oligonucleotide, a linker to a fluorophore, H, —OR^(c),—NR^(c)R^(d), —COOR^(c) or —CONR^(cR) ^(d) , wherein each R^(c) andR^(d) is selected from H, (C_(2-C) ₁₂)heteroalkyl,(C₃-C₁₂)heteroalkenyl, (C₃-C ₁₂)heteroalkynyl, (C₁-C₁₂)alkyl,(C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, aryl(C₁-C₁₂)alkyl and aryl, whereinone of R^(a) and R^(b) is a linker to the oligonucleotide or to afluorophore.
 12. The method of claim 2 wherein the inhibitor compositioncomprises one of the following structures:

wherein W is a linker having from about 3 to 100 main chain atoms,selected from C, O, N, S, P and Si; [A-B]_(n), is an oligonucleotidewherein A represents a sugar phosphate backbone, modified sugarphosphate backbone, locked nucleic acid backbone, peptidic backbone or avariant thereof used in nucleic acid preparation, wherein W is connectedto the 5′ end of [A-B]_(n), and wherein [A-B]_(n) comprises nucleotidescomprising ribofuranose having the following structure:

wherein Rz is —OCH₃, and B is a base, and wherein the nucleotides arelinked by natural phosphodiester linkages; n is an integer of from about3 to about 100; R_(a), R_(b), R_(c), R_(d), R_(e), and R_(f) aresubstituents selected from H, halogen, (C₁-C₈)alkyl, OR_(g), N(R_(g))₂,N⁺(R_(g))₃, SR_(g), COR_(g), CO₂R_(g), CON(R_(g))₂, (CH₂)_(m)SO₃ ⁻,(CH₂)_(m) CO₂ ⁻, (CH₂)_(m)OPO₃ ⁻², and NHC(O)(CH₂)_(m)CO₂ ⁻, and estersand salts thereof, wherein each R_(g) is independently H or(C₁-C₈)alkyl, and the subscript m is an integer of from 0 to 6; R_(h)and R_(w) are H or a group having from 1 to 30 atoms selected from C, N,0, P, and S and which is cyclic, acyclic, or a combination thereof. 13.The method of claim 6, wherein each ring in the MGB structure containsone or more additional substitutions selected from the group consistingof H, halogen, (C₁-C₈)alkyl, OR_(g), N(R_(g))₂, N⁺(R_(g))₃, SR_(g),COR_(g), CO₂R_(g), CON(R_(g))₂, (CH₂)_(x)SO₃ ⁻, (CH₂)CO_(x)CO₂ ⁻,(CH₂)_(x)OPO₃ ⁻², and NHC(O)(CH₂)_(x)CO₂ ⁻, AsO₃ ²⁻, and esters andsalts thereof, wherein each R_(g) is independently H or (C₁-C₈)alkyl,and the subscript x is an integer of from 0 to
 6. 14. The method ofclaim 11, wherein each ring in the MGB structure contains one or moreadditional substitutions selected from the group consisting of H,halogen, (C₁-C₈)alkyl, OR_(g), N(R_(g))₂, N⁺(R_(g))₃, SR_(g), COR_(g),CO₂R_(g), CON(R_(g))₂, (CH₂)_(x)SO₃ ⁻, (CH₂)_(x)CO₂ ⁻, (CH₂)_(x)OPO₃ ⁻²,and NHC(O)(CH₂)_(x)CO₂ ⁻, AsO₃ ²⁻, and esters and salts thereof, whereineach R_(g) is independently H or (C₁-C₈)alkyl, and the subscript x is aninteger of from 0 to
 6. 15. A method of treating a conditioncharacterized by over-expression of miRNA comprising administering aninhibitor composition to a subject at a concentration sufficient toinhibit the action of said miRNA, wherein the inhibitor compositioncomprises: a single-stranded oligonucleotide consisting of RNA; and aminor groove binder (MGB), wherein the oligonucleotide comprisesnucleotides, wherein all nucleotides of the oligonucleotide compriseribofuranose having the following structure:

wherein Rz is —OCH₃, and wherein B is a normal base or a modified base,wherein the nucleotides are linked by natural phosphodiester linkages.wherein the MGB is CDPI₃, wherein the MGB is conjugated to the 5′ end ofthe oligonucleotide, and wherein the miRNA is let-7 or miR-21.